3 Methods
3.1 Glycerolization
and Freezing of Red
Blood Cell
Concentrates
The starting materials in our process are leukocyte-reduced red
blood cell concentrates (RBCs) in SAG-M. The RBCs are prepared
according to the buffy coat method from 500 mL whole blood
donations after overnight hold at ambient temperature [28]. Briefly,
after a hard spin, the whole blood was separated in components
using an automated blood component separator (CompoMat, Fresenius HemoCare). RBCs were diluted with 110 mL SAG-M and
leukocyte reduced using the inline filter. The RBCs are glycerolized
and frozen shortly after production, preferably within 7 days after
collection (see Note 1). The RBC should be stored at 2–6
C in the
period between production and freezing. Before glycerolization,
the supernatant (additive solution) of the RBC is removed.
3.1.1 Removal
of Additive Solution
1. Sterilely connect a 150 mL transfer bag to the RBC bag, open
the weld, and transfer about 5 mL cell suspension to the
transfer bag. This will be used for quality control of the starting
product.
2. Weigh and record the weight of the unit. To obtain the net
weight, subtract the weight of the empty bag from the gross
weight.
3. Sterilely connect a new 150 mL transfer bag to the RBC bag.
This step will also disconnect the first 150 mL bag from the
RBC bag. Do not open the weld.
4. Place the RBC bag (and attached transfer bag) with the ports
upright in the bucket of the blood bag centrifuge, and fill the
bucket with water-filled bags to position the blood bag.
5. Weigh the filled bucket and pair it with a bucket with the same
weight (Æ2 g).
6. Spin the unit at 3200 Â g in a 22
C centrifuge for 5 min. To
minimize resuspension of the red blood cell-additive solution
interface, the brake should be set low.
7. Gently remove the unit from the centrifuge and place it in a
plasma extractor. Place a Kocher clamp on the tube between
the blood bag and transfer bag.
8. Open the weld. Open the Kocher clamp and express the supernatant fluid to the transfer bag. Replace the Kocher clamp on
the tubing between the two bags.
9. Detach the waste bag (containing supernatant) from the RBC
bag and discard.
10. Weigh and record the weight of the supernatant-reduced unit.
To obtain the net weight, subtract the weight of the empty bag
from the gross weight.
526
Johan W. Lagerberg
3.1 Glycerolization
and Freezing of Red
Blood Cell
Concentrates
The starting materials in our process are leukocyte-reduced red
blood cell concentrates (RBCs) in SAG-M. The RBCs are prepared
according to the buffy coat method from 500 mL whole blood
donations after overnight hold at ambient temperature [28]. Briefly,
after a hard spin, the whole blood was separated in components
using an automated blood component separator (CompoMat, Fresenius HemoCare). RBCs were diluted with 110 mL SAG-M and
leukocyte reduced using the inline filter. The RBCs are glycerolized
and frozen shortly after production, preferably within 7 days after
collection (see Note 1). The RBC should be stored at 2–6
C in the
period between production and freezing. Before glycerolization,
the supernatant (additive solution) of the RBC is removed.
3.1.1 Removal
of Additive Solution
1. Sterilely connect a 150 mL transfer bag to the RBC bag, open
the weld, and transfer about 5 mL cell suspension to the
transfer bag. This will be used for quality control of the starting
product.
2. Weigh and record the weight of the unit. To obtain the net
weight, subtract the weight of the empty bag from the gross
weight.
3. Sterilely connect a new 150 mL transfer bag to the RBC bag.
This step will also disconnect the first 150 mL bag from the
RBC bag. Do not open the weld.
4. Place the RBC bag (and attached transfer bag) with the ports
upright in the bucket of the blood bag centrifuge, and fill the
bucket with water-filled bags to position the blood bag.
5. Weigh the filled bucket and pair it with a bucket with the same
weight (Æ2 g).
6. Spin the unit at 3200 Â g in a 22
C centrifuge for 5 min. To
minimize resuspension of the red blood cell-additive solution
interface, the brake should be set low.
7. Gently remove the unit from the centrifuge and place it in a
plasma extractor. Place a Kocher clamp on the tube between
the blood bag and transfer bag.
8. Open the weld. Open the Kocher clamp and express the supernatant fluid to the transfer bag. Replace the Kocher clamp on
the tubing between the two bags.
9. Detach the waste bag (containing supernatant) from the RBC
bag and discard.
10. Weigh and record the weight of the supernatant-reduced unit.
To obtain the net weight, subtract the weight of the empty bag
from the gross weight.
526
Johan W. Lagerberg